An in vitro system has been developed in which F1 spleen cells generate cytotoxic activity directed against parental cells. The antigenic differences detected are controlled by the H-2D-Hh-1 region of the immune major histocompatibility complex. A number of positive correlations were demonstrated between hemopoietic graft rejection in vivo and F1 antiparent cell-mediated cytotoxicity in vitro. These include genetic polymorphism, immunologic maturation, antigen-induced specific unresponsiveness, and selective but nonspecific abrogation of responsiveness either by antisera or antimacrophage agents. The observations that F1 antiparent cytotoxic potential develops later than immunity to alloantigens and that selective abolition of F1 antiparent cytotoxicity can be achieved without abrogation of reactivity to alloantigens suggest that these two cell-mediated immune phenomena are generated by different mechanisms. The F1 antiparent cytotoxic system provides an in vitro model for elucidating the recognition and effector phases of hybrid resistance to parental hemopoietic grafts and may be useful in matching procedures for clinical bone marrow transplantation.